PROJECT SUMMARY The overall object of our research in liver ischemia reperfusion injury (IRI) is to comprehend mechanisms of inflammatory immune activation and resolution. In the last ten years, we have made significant progresses in dissecting molecular mechanisms of liver innate immune response against IR. However, its cellular basis is relatively less-well defined. Kupffer cells (KCs) are liver resident macrophages, which are highly heterogeneous and evolving kinetically in disease processes. Their response against IR has not been fully delineated, particularly in the context of recent finding that tissue resident and monocyte-derived infiltrating macrophages are distinctive in their lineages and functions. Our recent analysis of KC/macrophage subsets in IR livers reveals that liver inflammatory immune activation is associated with not only increases/activations of infiltrating macrophages (iMs), but also a drastic depletion of resident KCs (KCs). The opposite, i.e., contraction of iMs and recovery of KCs, is associated with the resolution of liver inflammation. Additionally, M1 polarization is evident in the activation stage, and M2 polarization in the resolution stage of liver immune response in both liver macrophage subsets. The purpose of this exploratory project is to perform proof of principle experiments to establish functional significance of KC depletion in liver IRI, and determine potential mechanisms of KC homeostatic functions in livers against IR. We hypothesize that IR triggers KC necroptosis which constitutes a key mechanism of liver inflammatory immune activation and functions in synergy with the infiltration and activation of iMs. Additionally, KCs execute the homeostatic function in liver IRI by phagocytosis of apoptotic/necrotic cells, which regulates KC innate immune activation and promotes their polarization towards immune regulatory/reparative type. We will test these two hypothesis in two specific aims. Our study will be the first to analyze specifically responses and functions of liver resident macrophages in IRI. Results will further our understanding of the disease pathogenesis, and potentially provide novel rationale to design appropriate cell-targeted therapies to ameliorate liver IRI.